Human cytomegalovirus (HCMV), a ubiquitous herpesvirus, is the leading infectious cause of birth abnormalities and a life-threatening pathogen in AIDS and organ transplantation patients. As one of the most complex animal viruses, the infectious virion is composed of a glycoprotein containing envelope, a tegument layer, and an icosahedral capsid enclosing a dsDNA genome. The HCMV virus and intermediate particles at different stages of assembly and maturation have been isolated from HCMV infected cells. These include the intranuclear A-, B-, C-capsids, the tegument containing cytoplasmic capsids, and the infectious and non-infectious virus particles. The proposed studies aim at understanding the structural basis and the mechanisms of HCMV capsid assembly, DNA encapsidation and tegument formation by determining the three-dimensional (3D) structures of HCMV virion, capsids and genetically-engineered viral particles using electron cryomicroscopy and computer processing. The 3D structures of wild-type virions, A-, B-, and C-capsids at 7-9 Angstrom units resolution will provide the framework of the molecular structures with visible alpha-helices for all subsequent structural studies. These relatively high resolution structures will make it possible to delineate structural changes associated with capsid morphogenesis and maturation. The 3D structural comparisons of the virion, detergent-treated de-enveloped virion particles and complexes with antibodies specific to the major tegument proteins will allow the identification of the tegument proteins and their binding sites on the capsid. Structural analyses of in vitro assembled capsids before and after proteolysis permit the visualization of the assembly protein/major capsid protein interactions. Non-icosahedral reconstructions of the virion capsids will provide the first structural information of the DNA packaging complex and may reveal the organization of the close-packed dsDNA. These studies represent our research efforts to establish a structural basis for HCMV infection and assembly. The long-term objective of this project is to understand the structural basis of HCMV capsid maturation and pathogenesis at high resolution and provide useful structural information for developing therapeutic strategies against HCMV infections.